Free Radical Scavenging Principles of Salvia reuterana Boiss. Aerial Parts

Salvia reuterana Boiss. is an aromatic perennial plant traditionally used for its anxiolytic and sedative properties. In the present study, various fractions and essential oil of S. reuterana aerial parts were investigated to find its free radical scavenging principles. Hydroalcoholic fraction with IC50 value of 112.6 ±3.2 μg mL-1 in DPPH assay demonstrated the highest free radical scavenging activity and was selected to further phytochemical investigation. RP-18 and Sephadex LH-20 column chromatography of the hydroalcoholic fraction resulted in the isolation and structural elucidation of four phenolic derivatives, including apigenin-7-O-β-D-glucopyranoside (1), luteolin-7-O-β-D-glucopyranoside (2), rosmarinic acid (3), and luteolin (4). Isolated compounds showed potent free radical scavenging activities (5.1-34.2 μg mL-1), compared with BHT (21.30 ± 1.9 μg mL-1). Twenty four compounds were also identified in GC-MS analysis of the plant essential oil, of which benzyl benzoate (26.64%), n-hexyl benzoate (22.99%) and n-hexyl isovalerate (6.04%) were the main compounds. The results of the present study introduced S. reuterana as a valuable source of natural phenolic antioxidants which can be utilized in prevention of oxidative stress related diseases. Moreover, interesting composition of S. reuterana essential oil, dominated by non-terpenes compounds (76.17%) especially aromatic derivatives, make it an appropriate candidate for more detailed studies.


Introduction
Nowadays, the role of free radicals, particularly reactive oxygen species (ROS), have been well recognized in development of many pathological disorders such as cardiovascular diseases, diabetes, cancers, inflammatory, and neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, etc.) (1). So, in recent years plants have received considerable attention as source of potent and safe natural free radical scavengers to prevent oxidative stress related diseases (2).
The genus Salvia L., commonly known as Sage, is one of the largest genera in the Lamiaceae family, mainly distributed in temperate and subtropical regions of the world (3). In the flora of Iran this genus is represented by 61 species, including Salvia reuterana Bioss. (4). This perennial aromatic plant is known as ''Maryam Goli-e Esfahani" in Persian and its flowering aerial parts are traditionally used in some parts of Iran as anti-depressant, as well as for the treatment of gastrointestinal disorders, eye pains and colds (5,6). Previous pharmacological studies have confirmed the anxiolytic (7), hypnotic (8), antidiabetic (9), antibacterial (10) and antioxidant (11)(12)(13) properties of S. reuterana aerial parts. In an comparative study, Esmaeili et al. reported that methanol extract of S. reuterana had the highest DPPH free radical scavenging activity (IC 50 ; 15.1 ± 1.00 µg mL -1 ) and ferric reducing power (FRAP value; 0.34 ± 0.01), among the other tested Salvia species (11). Methanol extract of the S. reuterana flowers has also been reported to possess higher free radical scavenging activity with (IC 50 ; 77.6 µg mL -1 ) in comparison with its leave extract (IC 50 ; 119.4 µg mL -1 ), in DPPH assay (12).
As a result of phytochemical studies on this plant aerial part, nine labdane diterpenoids with cytotoxic activity against HeLa and MCF-7 cell lines were isolated from its n-hexane extract (14,15). There are also some reports on essential oil composition of S. reuterana from different regions of Iran (10,12,16,17). Regarding the results of previous studies on considerable antioxidant activity of this medicinal species, the aim of the present research was the isolation and identification of the compounds involved in free radical scavenging activity of S. reuterana.

Plant material
The flowering aerial parts of S. reuterana were collected on Jun 2015 from the Khor region, Elburz province, Iran. A voucher specimen was deposited under the code 7045-TEH at the Herbarium of Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Extraction and fractionation
The air dried and grinded plant aerial parts (400 g) were macerated with 80% methanol in water (5 × 2.5 L). The total hydroalcoholic extract (82 g) was then dissolved in 500 mL of methanol-water mixture (8:2) and extracted by enough volumes of n-hexane and chloroform, successively, to get the n-hexane, chloroform and residual methanol-water (8:2) soluble (hydroalcoholic) main fractions.

Essential oil extraction
Hydrodistillation method using Clevenger apparatus was used to essential extraction from 100 g of dried and comminuted plant material for 3 hours. The pale yellowish oil was dried over anhydrous sodium sulphate and kept at 4 °C until analysis.
DPPH free radical scavenging assay Antioxidant activity of the total extract, main fractions and essential oil of plant aerial parts were evaluated by DPPH (2, 2-diphenyl-1-picryl-hydrazyl) free radical scavenging assay method described by Sarker et al. (18). Briefly, twofold serial dilutions (1.0 to 3.9×10 -3 mg mL -1 ) were made from samples, individually (each 2 mL). Two milliliter of freshly prepared DPPH (Sigma) solution (80 µg mL -1 ) was then added to each test tube. After 30 min, absorptions of the solutions were recorded at 517 nm using an Optizen 2120 UV PLUS spectrophotometer. Butylated hydroxy toluene (BHT) (Sigma), a commercial synthetic antioxidant, was also used as positive control. For each sample concentration causing a 50% reduction in absorption of DPPH solution (40 µg mL -1 ) was calculated as IC 50 . The experiment was repeated three times and results were expressed as Mean ± SD.

Isolation and purification of compounds
Hydroalcoholic fraction with the highest free radical scavenging activity (Table 1) was subjected to further phytochemical analysis. A portion of this fraction (4 g) was chromatographed on a reversed-phase (RP18, mesh 230-400, Fluka) column and eluted with the gradient mixture of methanol in water (0.5-9.5 to 7:3) to get five fraction (M1-5). Compounds 1 (18 mg) and 2 (43 mg) were isolated from the fraction M3 (320 mg) on a Sephadex LH-20 column (Fluka) eluted by methanol:water (8:2) as solvent system. Colum chromatography of the fraction M4 (147 mg) on a Sephadex LH-20 column with methanol resulted in the isolation of compound 3 (28 mg). Compound 4 (18 mg) was isolated from the fraction M5 (95 mg) through the reversed-phase column chromatography (methanol-water, 8:2). It was more purified on a Sephadex LH-20 column using methanol as eluent. All column chromatographies were monitored using thin layer chromatography (pre-coated silica gel 60 F-254 sheets, Merck) and the fractions giving same spots under 254 and 366 UV wavelengths were combined.

GC and GC-MS analysis
The plant essential oil was analyzed on an Agilent 7890B gas chromatograph with a DB-5 column (30 m × 250 μm id, 0.25 μm) connected to an Agilent 5977A mass selective detector (70 eV) under the following conditions; carrier gas: helium (1 mL min -1 ), temperature program: 50 °C for 5 min, 50-280 °C at 10 °C/min, injector temperature: 280 °C, injection volume: 1 μL. A homologous series of n-alkanes was also injected in conditions equal to the oil sample in order to calculate retention indices (RI). The compounds were identified by computer matching with the Wiley7n.L and NIST05a.L libraries, as well as by comparison of RIs and mass fragmentation patterns with those published in literature for standard compounds (19). GC-FID analysis of the oil was performed in the same conditions described above, for quantitative purposes.
A review on the results of the present study and previous reports shows a variation in essential oil composition of S. reuterana aerial parts collected from different regions of Iran (10,12,16,17). In an study by Fattahi et al. on essential oil analysis of seven wild population of S. reuterana from north and center of Iran, α-gurjunene (5.4-13.7%), β-elemene (4.5-13.9%), germacrene D (2.6-7.2%), spathulenol (1.0-8.0%) and n-hexyl acetate (1.2-6.8%) were identified as major compounds (16). Benzyl benzoate, the main compound of our analyzed essential oil sample (26.64%), has been detected in the range of trace to 8.0% in former mentioned study (16). n-hexyl benzoate (22.99%), another main compound identified in the present study was not detected by Fattahi et al. in their examined essential oils of different S. reuterana populations (16). However, n-hexyl benzoate has been characterized at high amounts (17.0%) in essential oil of S. reuterana flowers, collected from Kashan region, center of Iran (12). Benzyl benzoate and n-hexyl benzoate have also been reported in essential oil of Salvia multicaulis Vahl aerial parts with relative percentages of 60.3 and 16.7 (44). Differences in climate conditions, as well as possible presence of chemotypes in various S. reuterana populations are the factors which could be assumed as responsible for the observed variations in essential oils composition (45). However, a comprehensive study using more advanced chromatographic and spectroscopic techniques is needed for the assessment of variations between essential oil contents of different populations of S. reuterana.

Conclusion
The results of present study verify that S. reuterana with its potent free radical scavenging flavonoid and caffeic acid derivatives content (1)(2)(3)(4) can be considered as a valuable source of natural phenolic antioxidants. Literature review on biological activities of the isolated compounds also provides some molecular explanations for anxiolytic and antidiabetic properties, previously reported from S. reuterana. Moreover, interesting chemical composition of S. reuterana essential oils, which is dominated by non-terpenes compounds (76.17%), especially aromatic derivatives, make it an appropriate candidate for further studies.